Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase
| ISSN: |
0003-2697
|
|---|---|
| Source: |
Elsevier Journal Backfiles on ScienceDirect 1907 - 2002
|
| Topics: |
Biology
Chemistry and Pharmacology
|
| Type of Medium: |
Electronic Resource
|
| URL: |
| _version_ | 1798292448946946049 |
|---|---|
| autor | Cummings, J. Bartoszek, A. Smyth, J.F. |
| autorsonst | Cummings, J. Bartoszek, A. Smyth, J.F. |
| book_url | http://dx.doi.org/10.1016/0003-2697(91)90162-M |
| datenlieferant | nat_lic_papers |
| fussnote | An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-μm DEAE anion-exchange resin capable of chromatographing nucleic acids 〉 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 m potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [^1^4C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 m sodium chloride at 50^oC for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol100 μg), and the highest levels were recorded with oligonucleotides (243 pmol100 μg). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 μm [^1^4C]doxorubicin (18.6 pmol100 μg). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques. |
| hauptsatz | hsatz_simple |
| identnr | NLZ184067219 |
| issn | 0003-2697 |
| journal_name | Analytical Biochemistry |
| materialart | 1 |
| package_name | Elsevier |
| publikationsort | Amsterdam |
| publisher | Elsevier |
| reference | 194 (1991), S. 146-155 |
| search_space | articles |
| shingle_author_1 | Cummings, J. Bartoszek, A. Smyth, J.F. |
| shingle_author_2 | Cummings, J. Bartoszek, A. Smyth, J.F. |
| shingle_author_3 | Cummings, J. Bartoszek, A. Smyth, J.F. |
| shingle_author_4 | Cummings, J. Bartoszek, A. Smyth, J.F. |
| shingle_catch_all_1 | Cummings, J. Bartoszek, A. Smyth, J.F. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase 0003-2697 00032697 Elsevier |
| shingle_catch_all_2 | Cummings, J. Bartoszek, A. Smyth, J.F. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase 0003-2697 00032697 Elsevier |
| shingle_catch_all_3 | Cummings, J. Bartoszek, A. Smyth, J.F. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase 0003-2697 00032697 Elsevier |
| shingle_catch_all_4 | Cummings, J. Bartoszek, A. Smyth, J.F. Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase 0003-2697 00032697 Elsevier |
| shingle_title_1 | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase |
| shingle_title_2 | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase |
| shingle_title_3 | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase |
| shingle_title_4 | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase |
| sigel_instance_filter | dkfz geomar wilbert ipn albert fhp |
| source_archive | Elsevier Journal Backfiles on ScienceDirect 1907 - 2002 |
| timestamp | 2024-05-06T08:48:41.314Z |
| titel | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase |
| titel_suche | Determination of covalent binding to intact DNA, RNA, and oligonucleotides by intercalating anticancer drugs using high-performance liquid chromatography. Studies with doxorubicin and NADPH cytochrome P-450 reductase An HPLC method is described which can determine covalent binding to intact nucleic acid by intercalating anticancer drugs and at the same time remove noncovalently bound intercalated drug. The method uses a column containing a nonporous 2-μm DEAE anion-exchange resin capable of chromatographing nucleic acids 〉 50,000 bases in size in under 1 h. After priming with 1 mg of DNA, the column behaves as an intercalator affinity column, strongly retaining the drug while allowing the nucleic acid to pass through normally. Retained drug is released with an injection of 0.1 m potassium hydroxide. Incubations were performed with the intercalator doxorubicin, which is also believed to bind covalently to DNA. When [^1^4C]doxorubicin was mixed with DNA, at a concentration where all the drug would bind by intercalation, the column retained 82% of the total radioactivity, only 18% migrated with the nucleic acid. If the DNA was mildly denatured by treatment with 2 m sodium chloride at 50^oC for 45 min before chromatography, then 99.8% of total radioactivity was retained, only background counts migrated with the nucleic acid, as was the case with single-stranded DNA and RNA without any treatment. Purified NADPH cytochrome P-450 reductase was used to activate doxorubicin. DNA inhibited the metabolism of the drug by the enzyme, no covalent binding occurred with RNA, low levels occurred with single-stranded DNA (34 pmol100 μg), and the highest levels were recorded with oligonucleotides (243 pmol100 μg). The assay was sufficiently sensitive to measure covalent binding to DNA extracted from MCF-7 human breast cancer cells treated with 50 μm [^1^4C]doxorubicin (18.6 pmol100 μg). Thus, covalent binding to DNA, RNA, and oligonucleotides by intercalators can be measured quickly (20 min) without the need to either digest the nucleic acid or subject it to long sample preparation techniques. |
| topic | W V |
| uid | nat_lic_papers_NLZ184067219 |